Enzymes are unique in their ability to significantly enhance reaction rates under mild conditions with high levels of efficiency and selectivity. After substrates have bound to an enzyme's active site, their half-life is usually only a fraction of a second (Wolfenden, et al., Accounts of Chemical Research, 34, (12): 938-945 (2001)). Some enzymes perform catalysis so well they border on the physical limitations of efficiency with encountering substrate in solution, and have been known to achieve rate enhancements (kcat/kuncat) in excess of 19 orders of magnitude. These outstanding examples of enzymatic catalysis establish their remarkable power, and demonstrate their potential use in chemical synthesis.
In addition to rate enhancement, many enzymes are selective in their catalysis and allow for the direct production of single enantiomers. Single enantiomers of target product molecules have a large importance in the pharmaceutical and agricultural industries. Therapeutic compounds often act as structurally optimized inhibitors of biological processes. Since the human body functions using chiral chemistry, these compounds almost always contain chiral centers. In 2006, 80% of small-molecule drugs approved by FDA were chiral and 75% were single enantiomers (Thayer, Chemical and Engineering News, 85, (32): 11-19 (2007). USA Food and Drug Administration (FDA) regulations require proof that any non-therapeutic isomer comprising over 1% of the total composition be non-teratogenic. Thus, a racemic drug would require separate toxicology studies of each enantiomer. Further, as the complexity of these compounds increases, molecules often require more than one chiral center (Pollard, et al., Trends in Biotechnology, 25, (2): 66-73 (2007)). It is common for pharmaceutical processes to require a minimum acceptable enantiomeric excess (e.e.) of 98% to circumvent these difficulties.
Chiral intermediates are classically prepared by three different routes Carey, et al., Organic & Biomolecular Chemistry, 4, (12): 2337-2347 (2006)). One method is to simply attain them through a naturally occurring chiral synthon. This allows for an inexpensive and readily available source of natural products, but this pool of compounds is limited. Alternatively, chirality can be achieved through the resolution of a racemic mixture, as seen in the kinetic resolution of alcohols and amines through the application of lipases or esterases (Heine, et al., Protein Engineering Design and Selection, 20, (3): 125-131 (2007); Reetz, et al., CHIMIA International Journal for Chemistry, 50, (12): 668-669 (1996); Schmidt, Chembiochem, 7, (5): 805-809 (2006)). The major disadvantage of kinetic resolution is an unfavorable equilibrium constant, usually near unity. The use of a second reaction is required to drive the reaction to completion. Lastly, the chiral centers can be synthesized directly using an asymmetric catalyst or enzyme. If the desired selectivity is not achieved initially, multiple enrichments, such as crystallization, can be required to achieve an increased selectivity. In comparison to other catalysts, enzymes usually offer superior enantio- and regioselectivity, commonly reporting enantiomeric excesses “e.e.'s” of >99.9% (Bommarius, et al., Wiley-VCH Verlag GmbH & Co., (2004). High initial selectivity eliminates the need for enrichment, significantly reducing the cost of production.
One of the short comings of biocatalysis is the limited number of reactions which have identified enzymatic routes. In 2005, the American Chemical Society's (ACS) Green Chemistry Institute (GCI) and several leading global pharmaceutical companies created the ACS GCI Pharmaceutical Roundtable. The goals of this center encompassed innovation in the discovery, development, and production of pharmaceuticals. Consequently, the Roundtable identified the most aspirational reactions currently challenging the pharmaceutical industry (Constable, et al., Green Chemistry, 9, (5): 411-420 (2007). The reductive amination of a prochiral ketone with free ammonia to produce chiral amines ranked second on this list, and had yet to be achieved.
Therefore, it is an object of the invention to provide compositions and methods for reductive amination of a prochiral ketone to produce chiral amines.
It is another object to provide recombinant enzymes for the production of chiral compounds.